This proposal is a revision of a renewal proposal of GM59446, which has been funded for the period 08/01/03 to 07/31/07. Discoveries about the chemistry and biology of NO and its radical derivatives, especially HNO, continue to explode in the literature. Mechanistic understanding lags behind the experimental observations. Traditional tools of experimental study of mechanism are difficult to apply to these reactions, because they are often very fast, and a number of different reactive channels can, and often do, compete. Other times, especially in biological studies, the exact reactants or products are not known. This project has used the tools of computational quantum mechanisms to explore the mechanistic chemistry of nitrogen oxides and derivatives, especially NO, HNO, and RSNO. In the course of these studies, previously supported by the NIH, a variety of important fundamental discoveries have been made, such as the pKa of HNO, the reduction potential of NO, and improved estimates of the equilibrium constant for NO dimerization in an aromatic solvent. Mechanistic studies have led to an understanding of NO oxidation in air, various mechanisms of decompositions of RSNO, and how NO and HNO are generated from NONOates and Angeli's salt, respectively. Previously unrecognized reactive intermediates, nitroxyl disulfide, RSN(0)SR-, and thionitroxides, RSNHO*, have been discovered and characterized theoretically. The former has been validated experimentally. This new proposal builds on these discoveries and proposes to answer a variety of fundamental questions, such as whether thionitroxides can serve as readily reversible signals for protein processes commonly attributed to nitrosothiols, the nature of cysteine environments in proteins, how thionitroxides or nitrosothiols can be formed from thiols and NO, and what is the mechanism for the thermal uncatalyzed decomposition of nitrosothiols to disulfide plus NO. A number of critical questions involving the generation and role of HNO in biology, such as the mechanism of formation from nitrolipids, will be investigated. The success of the research described here can lead to new tools for study of physiological processes and drugs for hypertension, cardiac failure, and other biological processes involving NO metabolism.